Epigenetic programming of cancer-related inflammation byncRNA rheostat: Impact on tumor directed immune therapies

1. Amity Institute of Virology and Immunology, Amity University India 2. Department of Zoology, Central University of Punjab, Bathinda, Punjab, India 3. Department of Pathology/Lab Medicine, All India Institute of Medical Sciences Bathinda, Punjab , India 4. Department of Biochemistry, All India Institute of Medical Sciences, New Delhi, India 5. Laboratory Oncology Unit, Dr B R Ambedkar Institute Rotary Cancer Hospital, All India Institute of Medical Sciences, New Delhi , India


Inflammation: Bread and butter of Cancer
Inflammation is a non-specific host defence against tumor and is essential for both control and progression of cancer. This is manifested with the changes in Th1/2 (helper T cell 1/2) bias in patients' tumor micromilieu, which ultimately influences host immunity either in favour or against growing tumor mass in patients.
Immunogenic inflammation is pre-requisite for the host in controlling cancer growth mainly via tweaking M1 TAM (tumor associated macrophages), which promotes the immunogenic death of tumor cells by expression of interleukinsIL-2, IL-12 and IFN (Interferon) (Waldmann, 2018). On the other hand, when immunogenic inflammation persists, it becomes chronic and refractory where, it supports the growth of tumor mass by promoting in situ programming of tumor infiltrating M1 TAM towards M2 TAM, which is tropical and supports tumor cells development by promoting secretion of several proteases, cytokines and angiogenic growth factors (Yahaya et al., 2019) (Balahura et al., 2020).
Chronic inflammation is characterized by high levels of ROS (reactive oxygen species), which in concert with locally produced RNI (reactive nitrogen intermediates), promote DNA damage and trigger carcinogenesis (Broderick et al., 2015). Further, this chronic state is characterized by enhanced infiltration of immune effectorslike TAMs and T-regs (regulatory T cells), which secretecopious levels ofpro-inflammatory cytokines such as IL-1β, TNF-α (tumor necrosis factor alpha) andROS/RNS (reactive nitrogen species), which triggers fibrosis, excessive tissue damage,and tumorigenesis (Chiba et al., 2012).
Out of several factors which are present in tumor micromilieu,Th1 cytokines like IL-1β and IL-18 are major inflammatory cytokines which regulate cancer cell fate.IL-1β is responsible for Th17 programming ofimmune cells (Dinarello, 2009) [(Yasuda et al., 2019), which is decisive for the development of tumor. Typical Th17 response factors like TNF-α and IL-1 activate non-canonical NF-KB (nuclear factor kappa light chain enhancer of activated B cells) landscapewhich further enhances the secretion of IL-6, TNF-α, VEGF (vascular endothelial growth factor) and expression of iNOS (inducible nitric oxide synthase), that promotesinflammation, angiogenesis and tumor development (Dunn et al., 2012).However, in this line,the anti-cancer role of IL-18 is controversial; IL-18 induces the expression of IFN-γ, which is the central cytokine ofTh1 effector response and is decisive for immune-mediated destruction ofmany cancers (Esmailbeig and Ghaderi, 2017) (Zou et al., 2018).
A deeper analysis of molecular events has suggested inflammasomes as critical regulators of immunogenic inflammation in various infection-related aberrant immune pathologies. The Inflammasome is the bunch of intracellular proteins that get activated in response to PAMP (pathogen-associated molecular pattern) or DAMP (danger-associated molecular pattern) signalling and help maintain the homeostasis of the infected/damaged tissue in various stress conditions. NLRP12 (NOD-, LRRand pyrin domain-containing protein 12) is one of the major components ofthe Inflammasome,which negatively regulate the canonical NF-KB signalling pathway, control overt inflammation in hematopoietic and non-hematopoietic compartments (Allen et al., 2012). NLRP4 is expressed in many tissues and acts as a negative regulator of type-I IFN signalling and Autophagy (Jounai et al., 2011). It also inhibits IL-1β and TNF-α mediated NF-κB activation/signalling (Eibl et al., 2012) and is essential in mediating immune response against viral infections and cancer. In this direction, NLRP3 activation induces caspase-1 expression,which is decisive forthe secretion of IL-1β & IL-18 in active forms (Xue et al., 2019). These few examples corroborate the regulatory role of Inflammasome over immunogenic inflammation.
They suggest that these pathways may be involved in the polarization of either tumor infiltrating or intra-tumoral populations of M2-TAM, N2-TAN (tumor associated neutrophils) and T-regs.  (Chiba et al., 2012;Maiuri and O'Hagan, 2016), and are believed to contribute in tumorigenesis.

Non-coding RNAs: switch between inflammationcancer
As described above, acute inflammation often becomes chronic,especially in cancer or latent infection and work against the host. This is due to epigenetic changes in the host genome (Fardi et al., 2018). Thesechanges are instrumental in the plasticity of Non-coding RNAs supportcancer development in many ways, such as by (i) genetic alteration in the gene which codes for ncRNA (non-coding RNA) (Calin et al., 2002); (ii) amplification in chromosomal regions containing oncogenic ncRNA as seen inLncRNAs likeFAL1 (focally amplified LncRNA on chromosome 1)  and PVT1 (plasmacytoma variant translocation 1) (Tseng et al., 2014); (iii) stabilization of SNPs (single nucleotide polymorphisms) corresponding to LncRNAs H19 (Yuan et al., 2018) and CCAT2 (colon cancer-associated transcript-2) (Ling et al., 2013); (iv) inducing point mutations in the promoter region of ncRNAs like RMRP(ribonuclease mitochondrial RNA processing) and NEAT1(nuclear enriched abundant transcript 1) in breast cancer (Rheinbay et al., 2017); (v) dysregulating the synthesis of an enzyme such as Drosha and Dicer which are involved in the processing of miRNA (Rupaimoole and Slack, 2017). Besides this, some LncRNA like NKILA(NF-KappaB Interacting LncRNA) has dual roles, NKILAcan negatively regulate canonical NF-KB signalling, immunogenic inflammation, and cancer. In contrast, NKILA is also capable of promoting tumor development by mitigating TH1 response and CTL inactivation  and promote tumor development. Given this, temporal regulation of ncRNAs in the host is believed to control tumor development or their sensitivity for tumor directed interventionsthrough transcriptional, post-transcriptional and epigenetic processes (Adams et al., 2014;Anastasiadou et al., 2018;Rupaimoole and Slack, 2017). Further an overview of role of non-coding RNAs in controlling and promoting cancer development are discussed inTable 1&2 and depicted in

MiRNAs: Internal bottlenecks
miRNAs are non-coding molecules that are detrimental in chronic inflammation for promoting cancer in the host. For example, miRNA-223blocks IL-1β production by blocking 3'-UTR of NLRP3 mRNA and suppresses NF-KB signalling for the progression of squamous cell carcinoma (Yan et al., 2019b). In contrast, the expression of miR-133a-1 in the host increases the activity of NLRP3/caspase-1 and active IL-1β production (Bandyopadhyay et al., 2013). Whereas, miR-21 deficiency can inhibit NLRP3 activation and IL-1β & caspase-1 expression (Xue et al., 2019), and help host immunity in controlling tumor growth more effectively.
Cancer cells rely upon many signalling events that enable them to evade immune attacks mainly by subverting immunogenic inflammation that is hostile to their existence. In this context, non-canonicalNF-kB signalling is known to trigger tumorigenesis (Chew et al., 2018), which is primarily manifested by miR-146which targetsIRAK1(Interleukin-1 receptor-associated kinase 1) and TRAF (TNF receptorrelated factor) and in-turn controls NF-kB (Taganov et al., 2006)  Notch1 (Notch homolog 1) or Jag1 (Jagged1) pathways,as seen in endometrial cancer (Zhou et al., 2018). Compelling studies suggest that inhibiting the expression of LncRNA-P21 promotes M1 polarization  and reduces tumor development.Paradoxically LINC00662 promotes WNT3A secretion by tumor cells which drive M2 polarization via the Wnt/β-catenin axis .

Conclusion and major perspective
By utilizing the knowledge of non-coding RNA, Personalizeds immunotherapy can harness many benefits from currently available techniques like RNA destabilizing elements (RDEs) like poly(A) signals; blocking LncRNA promoter; transcript degrading; transcript silencing; inhibitors which can interfere with LncRNA interaction with the target; aptamers; gene editing/regulating tools like CRISPR, CRISPRi, and CRISPRa; and synthetic mimics (Zhang et al., 2020b). This knowledge can be currently used at its best in combinational cancer immunotherapy regimes like immune checkpoint blockade (ICB) therapy and chimeric antigen receptor T cell therapy (CAR-T). Integrating these tools with the findings as discussed above can yield very novel and specialized therapeutic interventions. For example, in a mice study, LncRNA-NKILA, which turns T cell sensitive to AICD, was targeted with shRNA showed positive results, overcoming immune evasion and AICD . Similarly, LncRNA-INCR1 increased the cancer cell susceptibility to cytotoxic T cells and increased the efficacy of CAR-T treatment by controlling IFN-γ signaling (Mineo et al., 2020).
However, there are several limitations in this direction, as the difficulties with specific delivery of these molecules, with nano-particle mediated delivery and exosomemediated delivery as the only options available. Besides this, the unstable structure, poorly conserved nature, which means the animal studies developments cannot be implemented on humans, and unknown side effects of LncRNA contribute to major roadblocks (Zhang et al., 2020b). So far, the knowledge we have in this direction is very naïve and preliminary, therefore seeks answers to many questions. However, LncRNA can be considered for combinational therapy with drugs and therapeutic antibodies used in cancer therapy (Sharma et al., 2020). Using/targeting these LncRNA according to their pro-cancer/anti-cancer roles can potentially manipulate the sensitivity of cancer towards chemotherapeutic drugs , radiotherapy, and monoclonal antibody (Wang et al., 2019b).